2-Aryl-7-substituted pyrazolo {8 1,5{i a{b {9 1,3,5-triazines

ABSTRACT

2-ARYL-7-SUBSTITUTED PYRAZOLO(1,5A)1,3,5-TRIAZINE DERIVATIVES ARE DISCLOSED WHICH ARE USEFUL AS 3&#39;&#39;,5&#39;&#39;-CYCLIC AMP phosphodiesterase inhibitors. These compounds are prepared by condensation of aryl- Beta -ketonitrile derivatives with thiosemicarbazide which affords the corresponding 3-amino-2thiocarbamoyl-5-aryl pyrazoles which, when heated with diethoxymethylacetate, give 2-aryl-7-mercapto pyrazolo (1,5a)1,3,5-triazines. The mercapto derivatives may then be methylated with methyl iodide to afford 2-aryl-7-methylthiopyrazolo(1,5a)1,3,5,-triazines. Nucleophilic displacement of the methylthio moiety with various amines and sodium alkoxides yields the appropriately substituted 2-aryl-7-substituted pyrazolo(1,5a)1,3,5-triazines.

United States Patent Kobe et al.

2-ARYL-7-SUBSTITUTED PYRAZOLO [1,541] 1,3,5-TRIAZINES Inventors: Joze Kobe, Costa Mesa; Darrell E. OBrien, Mission Viejo; Roland K. Robins, Santa Ana. all of Calif.

ICN Pharmaceuticals, Inc., Pasadena, Calif.

Assignee:

U.S. Cl 260/248 NS, 260/249.5, 424/249 Int. Cl C07d 57/34 Field of Search 260/248 NS, 249.5

References Cited UNITED STATES PATENTS 12/1970 Lewis et a] 260/2495 Primary Examinef-John Ford Attorney, Agent, or Firm-Lyon 8L Lyon [57] ABSTRACT 2-aryl-7-substituted pyrazolol 1,511] l ,3,5-triazine derivatives are disclosed which are useful as 3'.5'-cyclic AMP phosphodiesterase inhibitors. These compounds are prepared by condensation of aryl-B-ketonitrile derivatives with thiosemicarbazide which affords the corresponding 3-amino-2-thiocarbamoyl-5-aryl pyrazoles which, when heated with diethoxymethylacetate. give 2-aryl-7-mercapto pyrazolo [l.5a]'l.3,5-triazines. The mercapto derivatives may then be methylated with methyl iodide to afford 2-aryl-7-methylthiopyrazolol 1,5a1l ,3.5,-triazines. Nucleophilic displacement of the methylthio moiety with various amines and sodium alkoxides yields the appropriately substituted 2-aryl-7-substituted pyrazolo[ 1,5a] l,3,5- triazines.

11 Claims, No Drawings 2-ARYL-7-SUBSTITUTED PYRAZOLO 1,5,4

.. -l-TRIMINES. 7

BACKGROUND OF THE INVENTION cells of the hormone. Cyclic AMP has thus been shown to activate protein kinases, which in turn produce physiological effects such as muscle contraction, glycogenolysis, steriodogenisis and lipolysis.

Cyclic AMP is degraded, however, in vivo by phosphodiesterase enzymes, which catalyze hydrolysis of the cyclic purine nucleotide to '-adenosine monophosphate with a consequent loss of function. It has accordingly been suggested that substituted cyclic AMP analogs whichare more resistant to phosphodiesterase degradation than the naturally occurring cyclic nucleotide might be administered in aid of lagging cellular processes. Synthetic production of such compounds, however, is quite costly. It would be advantageous, therefore, to enhance the beneficial effects of naturally produced cyclic AMP by administering compounds which are capable of inhibiting the undesirable effects of phosphodiesterase enzymes.

Sutherland, et al., in Circulation 37,279 (1968), suggest that thepharm'acological effects of theophylline, which has the structure are the result of its ability to inhibit the action of phosphodiesterase enzymes. Theophylline has thus been employed in lieu of the adenyl cyclase-stimulating hormones, epinephrine and norepinephrine, as a heart stimulant following-cardiac arrest and in refractory asthma cases as a bronchial dilator. Theophylline, however, does not selectively inhibit phosphodiesterase, but rather gives general stimulation to the central nervous system. Accordingly, the use of theophylline can make the recipient nervous and irritable and can also create cardiovascular effects, i.e., rapid beating. By the same token, theophylline is not as potent as a phosphodiesterase inhibitor as is desired and consequently has to be used in larger quantities, which, or course, can further the undesirable effects enumerated above.

F. L. Rose et al in articles appearing in J. Chem. 800., 5642 (1963); 3357 (1965) and 1593 (1969) have reported a number of triazolo[2,3clpyrimidines a'nd triazolo[4,3c]pyrazines (for example, compounds I and 2 below), which are somewhat structurally related to theophylline and reportedly capable of protecting animals from histamine-induced bronchospasm.

. H N mym Z u s 7 a As indicated, however, in the application of Darrell E. OBrien, et al., Ser. No. 206,538, entitled 3,5,7- trisubstituted pyrazolo[l,5a]pyrim.idines, assigned to the same assignee as this application, evaluation of compound I to determine its phosphodiesterase inhibition capability showed that compound to be approximately equal in effect to theophylline with phosphodiesterase enzyme isolated from rabbit lung, but only about 0.2 as effective with phosphodiesterase enzyme obtained from rabbit kidney. Also, as set forth in such OBrien, et al., application, the defined trisubstituted pyrazolo[ l,5a] pyrimidines have been found to possess phosphodiesterase inhibition properties. In view of the clear need, however, for phosphodiesterase inhibiting compounds of significant potency and capable of providing the previously discussed selective inhibitions, we have discovered that 2-aryl-7-substituted pyrazolo[l- ,5a]l,3,5 -triazines, as defined hereinafter, also possess phosphodiesterase inhibitory capability.

SUMMARY OF THE INVENTION According to this invention, there are provided phosphodiesterase inhibitors of the following structure fiEr wherein R is SH; NH OH; NI-I--NH N=N=N; SR 0R NHR, or N(R,) where R, is a C,C alkyl; or

and Ar is an aryl group such as phenyl or a substituted phenyl derivative.

DETAILED DESCRIPTION OF THE INVENTION used as starting materials, with cyclization of each of such intermediates with diethoxymethylacetate giving the corresponding 7-hydroxy (compound D), 7- mercapto (compound E), and 7-amino (compound F) derivatives of Z-phenylpyrazolo [l,5a] 1,3,5-triazine. The mercapto derivative (compound E) may be methylated with methyl iodide in a sodium hydroxide solution to afford 7-methylthio-2-phenylpyrazolo[ 1 ,Sa] 1 ,3,5- triazine (compound G). The methylthio group is very reactive toward nucleophilic reactions and by treatment with a sodium hydroxide solution or ammonium hydroxide the 7-hydroxy (compound D) and 7-amino (compound F) derivatives respectively are obtained. Analogously, when compound G is stirred at room temperature with hydrazine hydrate, the 7-hydrazino-2- phenylpyrazolo[ l,5a]l,3,5-triazine (compound H) is obtained.

Treatment of the 7-hydrazino derivative (compound H) with nitrous acid affords a mixture of the azido and tetrazolo derivatives (compound I). The infra-red spectra (KBr) indicates that only the azido structure is present and the nuclear magnetic resonance spectra in trifluoroacetic acid indicates only the azidoisomer. The nuclear magnetic resonance spectra in DMSO, however, shows that tha azido and tetrazolo isomers are present in a ratio of 1:3. Compound H may also be converted to the cyclized product (compound J) with the diethoxymethylacetate at room temperature. Compound is quite stable under normal conditions, although rearrangement to the isomeric compound K has been observed under more severe conditions. Analogously, the reaction of compound G with primary and secondary amines gives 2-phenyl-7-substituted amino pyrazolo[ 1 ,Sa] 1,3,5-triazine derivatives, compounds L, M, N and O.

W-K a Z a/ N low. In all such examples, parts and percentages are by weight and temperatures in degress Centigrade unless otherwise indicated. Melting points were taken on a Thomas-Hoover melting point apparatus and are uncorrected. lnfra-red nuclear resonance spectra were determined on a Perkin-Elmer 257 grating infra-red spectrophotometer and on a Hitachi Perkin-Elmer R-20A high resolution nuclear magnetic resonance spectrophotometer respectively. All samples displayed a single spot on thin layer chromatography and were analyzed by the Heterocyclic Chemical Corporation of Harrisonville, Miss.

EXAMPLE l ps 2-phenylpyrazolo[ 1 ,5a] 1 ,3,5-triazin-7[4Hl-thione (compound E) EXAMPLE ll 7-methylthio-2-phenylpyrazolo[1,5a]l,3,5-triazine (compound 0) Sodium hydroxide (100 mg) was added to a solution of compound E (550 mg., 2.5 mmoles) in 15 ml of 33 percent ethanol. Methyliodide (600 mg) was added dropwise to this solution and after a short period of stirring, the white product began to separate. After stirring The invention will be further understood by reference to the specific but illustrative examples which folfor 30 minutes at room temperature the mixture was evaporated to half-volume at reduced pressure. The

white product was separated by filtration. The white product (500 mg, 75 percent) was recrystallized from a mixture of ethylacetate hexane to afford an analytically pure product, MP 140-2.

Anal. Calcd. for C H N S: C, 59.5; H, 4.16; N, 23.]. Found: C, 59.9; H, 4.47; N, 23.4.

EXAMPLE lll 2-pheny1pyrazolo[1,5a]1,3,5-triazin-7[4H]-one (compound D) Method A *A mixture of compound Gin ml of 1N NaOH solution and 5 ml of ethanol was stirred at room temperature for 24 hours. At the end of this time the solution was acidified with 2N H SO and the precipitated product was separated by filtration. The product weighed 400 mg (66 percent) and was purified by recrystallization from a mixture of ethanol and water to afford an analytically pure product that had a MP of 258-60.

Anal. Calcd. for C,,H,,N O: C, 62.2; H, 3.80; N, 26.4. Found: C, 62.2; H, 3.77; N, 26.4.

Method B A mixture of compound A (370 mg) and diethoxymethyl acetate (1.25g) was heated at reflux. The product which separates from the hot reaction mixture (300 mg, 78 percent) was identical in all respects to the product obtained by Method A.

EXAMPLE 1V 7-amino-2-pheny1pyrazolo[1,5a]1,3,5-triazine (compound F) Method A A solution of compound C (750 mg, 3.7 mmoles) in diethoxymethyl acetate (3.5 g) was heated at reflux for 5 minutes. The solution was evaporated to dryness and the residue recrystallized from a mixture of ethanol and water to affored 750 mg (80 percent) of analytically pure product. MP 283-6.

Anal. Calcd. for C H,,N,,: N, 33.2 Found: N, 33.1.

Method B A mixture of compound (i (750 mg, 3.1 mmoles), ethanol ml), and ammonium hydroxide solution (28 percent) (0.7 ml) was heated at 100 in a stainless steel pressure bomb. After 16 hrs., the mixture was evaporated to dryness and the residue was recrystallized from a mixture of ethanol and water to yeild 500 mg of analytically pure product.

EXAMPLE V EXAMPLE V1 2-phenyl-s-triazolo[3,4glpyrazolol 1,5011 ,3,5-triazine (compound .1)

A suspension of compound H (l.0 g, 4.4 mmoles) in diethoxymethylacetate (5.0 g) was stirred at room temperature for 1 hr. The precipitated produce was separated by filtration and recrystallized from a mixture of dimethylformamide and toluene to yield 1.0 g (90 percent) of analytically pure product. MP 2635.

Anal. Calcd. for C H N C, 61.0; H, 3.41; N. 35.6. Found: C, 60.9; H, 3.18; N, 35.3.

EXAMPLE Vll 2-phenyl-s-triazolol3.2g1pyrazo1ofl1,5a]1,3,5'triazine (compound K) Compound J (250 mg) was heated at its melting point for 5 minutes. The melt was allowed to solidify and then recrystallized from ethylacetate to afford 200 mg percent) of analytically pure product that had a melting point of 213-5.

Anal. Calcd. for C H N C, 61.0; H, 3.41; N, 35.6. Found: C, 61.0; H, 3.37; N, 35.5.

EXAMPLE Vlll The reaction of 7-hydrazino-2-phenylpyrazolol1 ,Sa] 1,3,5-triazine (compound H) with sodium nitrite to afford compound 1.

A solution of compound H (270 mg, 1.2 mmoles) in 5 ml of 2N hydrochloric acid was cooled to 10 and a solution of soidum nitrite (120 mg) in water (10 ml) was added dropwise. After the addition was complete the mixture was stirred at room temperature for 2 hrs.

The product was separated by filtration, washed with water and recrystallized from methanol to afford 250 mg percent) of analytically pure product. MP 188-90.

Anal. Calcd. for C H N C, 55.7; H, 2.97; N, 41.3. Found: C, 55.4; H, 3.02; N, 41.5

The infra-red spectra (KBr) shows a strong azido band at 2,150 cm and a strong absorption at 1,182 cm". This unassigned band, which is not present in the other derivatives of Z-phenylpyrazolol1,5a]1,3,5- triazine, could possibly be the tetrazolo ring absorption as reported by Avramenko, et al., Khimiya Gelcmlsikliclwskikh Soedinenii, 4, 423 (1968). The nmr spectrum (DMSO) shows both the azido and tetrazolo isomers. The quilibrium constant K [(C azido)/(tetrazo1o] is K 37 0.5 and K 70 1.5. The nmr spectra (trifluoroacetic acid) shows only the expected azido structure.

A solution of the product mg) in xylene (5 ml) was refluxed for 98 hours and then cooled. The white solid (50 mg) that separated was identical in all respects to 7-amino-2-phenylpyrazolo[1,5a]1,3,5- triazine (compound F).

Although the compounds of this invention have been thus far described as phenyl triazine derivatives, as previously indicated, Ar may be other aryl groups such as a substituted phenyl derivative, as for example, halogenated, alkylated (C to C or a methoxy substituted derivative. Such compounds may be prepared following the procedure previously described, utilizing, of course, the appropriate aryl ketonitrile derivatives as starting materials.

15X AMPLE IX 2-phcny1-7-n-propylaminopyrazolol l,5a]1,3,5-triazine (compound L) A solution of 7-methylthio-Z-phenylpyrazolo[1.5a] 1,3,5-triazine (G) (1.21 g, 5 mmoles) and npropylamine (0.30 g, 5 mmoles) in 50 ml of anhydrous methanol was stirred at room temperature for 2 days (or refluxed for 4 hrs). At the end of this time the solution was evaporated to dryness. The residue was recrystallized from a mixture of ethanol and water to afford 1.2 g (96 percent) of analytically pure material that had a melting point of l40l.

Anal. Calcd. for C H NQC, 66.4; H, 5.97; N, 27.7. Found: C, 66.4; H, 6.08; N, 27.9.

EXAMPLE x 7diethylamino-2-phenylpyrazolo[ 1,5a] 1,3,5-triazine (compound N) A mixture of 7-methylthio-2-phenylpyrazolo[1,50] 1,3,5-triazine (G) (0.8 g, 3.3 mmoles) and diethylamine (0.24 g, 3.3 mmoles) in 25 ml of absolute ethanol was heated at reflux for 36 hrs.- At the end of this time, the solvent was removed at reduced pressure and the dark oil residue was covered with 10 ml of water. The white solid residue that precipitates was separated by filtration and washed with an additional 10 ml of water. Recrystallization from n-heptane afforded 0.6 g (71 percent) of analytically pure material that had a melting point of 878.

Anal. Calcd. for C, H N C, 67.4; H, 6.41; N, 26.2. Found: C, 67.40 H, 6.44 N, 26.30

EXAMPLE Xl Z-phenyl-7-piperidinopyrazolol 1,541] l ,3,5-triazine (compound A solution of 7-methylthio-2-phenylpyrazolol1.5a] 1,3,5-triazine (G) (1.5 g, 6.9 mmoles) and piperidine (0.59 g, 6.8 mmoles) in 50 ml of anhydrous methanol was heated at reflux for 8 hrs. At the end of this time, the solvent was removed at reduced pressure and the residue titurated with 10 ml of water. The residue was recrystallized from a mixture of methanol and water to afford 0.97 g (50) of analytically pure product that had a melting point of 82-4.

Anal. Calcd. for C H N C, 68.8; H, 6.13; N, 25.1. Found: C, 68.75; H, 6.12; N, 25.29.

7' EXAMPLE xn 7-n-butylamino-2-phenylpyrazolo[1.5a]1,3,5-triazine (compound M) A solution of 7-methylthio-2-phenylpyrazolo[1,5a] 1,3,5-triazine (G) (1.5 g, 6.9 mmoles) and nbutylamine (0.51 g, 6.9 mmoles) in 50 ml of anhydrous methanol was refluxed for 4 hrs. At the end of this time, the solvent was removed at reduced pressure and the residue titurated with ml of water. The crystalline residue was recrystallized from a mixture of ethanol and water to afford 1.6 g (94 percent) of analytically pure product that had a melting point of l20-2.

Anal. Calcd. for C H N C, 67.4; H, 6.41; N, 26.2. Found: C, 67.05; H, 6.78; N, 26.58.

EXAMPLE Xlll The compounds of this invention have been tested for their ability to inhibit action of the enzyme phosphodiesterase.

3,5'-cyclic AMP phosphodiesterase (PDE) has been isolated and purified from two different tissues in the following manner. Homogenates of rabbit heart and rabbit lung were made in sucrose-Tris-magnesium buffer and were subjected to centrifugation at low speed to remove nuclei and cell debris. The supernatants were then centrifuged at 105,000x g for 30 minutes. The 105,000x g supernatants were then fractionated using (NH SO.,. The precipitation which formed at 0-30 percent saturation was collected by centrifugation at 20,000x g and dissolved in Tris-magnesium buffer and dialyzed overnight against the same buffer. A second (NH SO fraction was obtained by raising the concentration of the first supernatant to 50 percent. These two (NH SO fractions as well as the supernatant from the 30-50 percent out were then assayed for PDE activity using the method of Appleman, Biochemislry 10, 31 1 (1971). The first fraction obtained from both heart and lung tissue was found to contain a PDE with low affinity for 3', 5-cyclic AMP (high Km). The second fraction was found to exhibit a biphasic curve when the Lineweaver-Burk method of analysis was used. This indicates either the presence of two separate enzymes, one having a high and the other a low affinity for the enzymes, or one protein with two separate sites. Appleman, supra, indicates that extracts of brain yield two separate enzymes (a high Km and a low Km) which can be separated by sepharose gel chromatography.

All of the inhibitory studies reported here were performed with the high affinity (Fraction [1, low Km) enzyme obtained from rabbit heart or rabbit lung. 1 values were calculated in some instances from a plot of log 1 vs. percent 1 in experiments in which inhibitor concentration was varied over a wide range, at a constant 3', Sf-cyclic AMP concentration of approximately 1.7 x 10 M. The relative inhibitory activity of each compound is compared with theophylline is expressed as an a value. This value is obtained by dividing the 1 value for theophyllinein a particular experiment by the 1 value obtained for the particular compound being evaluated. in most instances a values were calculated from an inhibition study performed with a single concentration of test compound as long as the inhibition produced by that concentration was from 20-80 percent. ln this instance an a value was calculated by dividing the c dn centration of test substance giving x percent inhibition/concentration of theophylline giving the same (X percent) inhibition.

The validity of this method has been checked by comparing a values obtained by (1) measurements at a single concentration of inhibitor and (2) measurements at four concentrations of inhibitor (I50 determinations). a values compared in this way have been found to agree to within 10 percent of each other.

The basincubation fixtfie coiitained the following substances (amounts in umoles): H-cAMP (specific activity -2,l cmp/pmole), 0.00016; Tris pH 7.5, 40;

MgCl 0.5; Enzyme (cAMP phosphodiesterase), 5-50 ,ug protein; and 10' to 10 molar concentration of the inhibitor; incubation time 10 minutes at 30C. At the end of incubation the mixtures are heated to C for 2 minutes and ug of snake venom phosphodiesterase from Crotalus atrox was added and the tubes incubated for 10 minutes at 30C. The mixture was then cooled and 1 ml of a Dowex l2X, 200-400 mesh suspension, prepared by mixing 100 g of the resin in 200 g H O, was added and the mixture centrifuged. An aliquot of the supernatant was used to determine counts per minute using a liquid scintillation spectrometer.

9 10 Zero time values were obtained using incubations in compounds possess selective phosphodiesteruse enwhich the CAMP phosphodiesterase was omitted from zyme inhibitory capability. the first incubation. The '/-hydrazino-2-phenyl-pyrazolo[ l,5a]1,3.5-

" puma 1 INHIBITION OF 3',5'-CYCLIC AMP PHOSPHODIESTERAISE ISOLATED FROM RABBIT LUNG AND HEART M\N th Compound Ar R ulung aheart D phenyl on 0.6 0.4

1: phenyl Sll 3.2 3.5

F phenyl NH 3.0 2 0 G phenyl SCH 11.0 7 .3

ll phenyl Nll-Nl'l 0.6 l 0 L phenyl l lllCll -Cll -Cll 26.0 3.8

M phenyl Nil-CH -Cll -CH -Cll 13.5 2.5

N phenyl N(Cli2-Cil3)2 143 S7 0 phenyl 29 11 M J F It will be observed from the foregoing results that triazine has been tested in vivo and shown ability to inseveral of the compounds of this invention have shown hibit death in mice from anaphylactic shock at a coninhibition capability significantly superior to theophyl- 6O centration of 100 mg/kg. line, with 2-phenyl-7-diethylamino pyrazolo[ I- We claim: ,5a]l,3,5-triazine (compound N) demonstrating vastly 1. A compound of the structure superior capability, 143 times greater for rabbit lung phosphodiesterase and 57 times greater for rabbit heart N phosphodiesterase. The other 7-alkylamino and piperi- 65 r A dino substituted compounds likewise showed inhibition M N\ r capability far superior to theophylline. By the same to- Y ken, the results shown in Table I indicate that these Q and wherein Ar is phenyl or phenyl substituted with halogen, C to C alkyl or methoxy.

2. The compound of claim 1 in which Ar is phcnyL 3. The compound of claim 2 in which Ar is phenyl and R is NH 4. The compound of claim 2 in which Ar is phenyl and R is an alkyl amine.

5. The compound of claim 2 in which Ar is phenyl and R is 6. The compound ofcluim 4 in which R is N(R and R is C H 7. 2phenyl-7-diethylumino pyrazololljalljj triuzinc.

8. 2-phenyl 7-pipcridinopyruzolol 1,50 l ,3 5 l0 triuzinc.

9. 2-phenyl-7-n-propylaminopyrazolol I .511] l 3.5- tritizine.

l0. 2-phenyl-7-n-butylaminopyruzolol 1,5a] 1.35- triazine.

ll. 2-phenyl-7-methylthi0pyrazol0[ 1,5a] l ,3,5- triazine. 

1. A COMPOUND OF THE STRUCTURE
 2. The compound of claim 1 in which Ar is phenyl.
 3. The compound of claim 2 in which Ar is phenyl and R is NH2.
 4. The compound of claim 2 in which Ar is phenyl and R is an alkyl amine.
 5. The compound of claim 2 in which Ar is phenyl and R is
 6. The compound of claim 4 in which R is N(R1)2 and R1 is C2H5.
 7. 2-phenyl-7-diethylamino pyrazolo(1,5a)1,3,5-triazine.
 8. 2-phenyl-7-piperidinopyrazolo(1,5a)1,3,5-triazine.
 9. 2-phenyl-7-n-propylaminopyrazolo(1,5a)1,3,5-triazine.
 10. 2-phenyl-7-n-butylaminopyrazolo(1,5a)1,3,5-triazine.
 11. 2-phenyl-7-methylthiopyrazolo(1,5a)1,3,5-triazine. 